The goal of this research program is to elucidate physical mechanisms of gene control processes mediated by the interactions of proteins at specific sites on DNA. Components of the program include: (a) development of new techniques for the study of site-specific protein-DNA interactions, (b) experimental studies of functional energetics in specific systems, (c) correlation of the energetic properties with structural features of the interacting molecules, and (d) development and testing of physical- chemical models for gene control. We plan to extend our previous studies on the repressor-operator control system of bacteriophage lambda, which is a prototype for a large family of cooperative multi-site gene control systems. Using techniques of calorimetry in combination with quantitative DNase footprint titration and a new cryogenic gel shift technique that we have developed, we will carry out a detailed study of the effects of temperature, pH, and salt interactions on the site-specific energetics (including cooperativity effects) of cI and cro repressor binding to the DNA operator sites. The results will provide new insights into the detailed roles of proton binding, salt binding, and the various non-covalent forces responsible for the regulatory interactions. These studies will be extended to a series of mutant repressors that are deficient in cooperativity. We will study the biological role of cooperative interactions between operator-bound repressors by in vitro and in vivo studies of mutant repressors that have altered cooperativity, using a physical-chemical model of gene control that is being developed under this program of research.